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Objective‍ ‍To map the genome-wide distribution profile of histone H3K27me3 modification in diffuse gastric cancer tissues， identify target genes regulated by H3K27me3， and primarily explore the potential mechanism of its modification reprogramming in the occurrence and development of the tumor. Methods‍ ‍Normal gastric mucosal tissues and diffuse gastric cancer tissues were harvested from the patients who underwent examinations or treatments in the departments of gastroenterology and gastrointestinal surgery of our medical center between 2021 and 2023. There were 14 patients in the normal group （6 males and 8 females， average age of 46 years） and 14 patients in the gastric cancer group （8 males and 6 females， average age of 63 years）. Cleavage under target and tagmentation （CUT&Tag） technology was employed to capture genomic regions modified by H3K27me3， and analyze the reprogramming characteristics of these modifications. RNA sequencing data， data from high-throughput chromosome conformation capture （Hi-C） technology， and publicly available single-cell data were integrated to investigate the target genes regulated by the reprogramming of H3K27me3 modifications in diffuse gastric cancer cells. Results‍ ‍The quality of the CUT&Tag and RNA sequencing data met the standards required for subsequent analysis. Histone H3K27me3 modifications in normal gastric mucosa and diffuse gastric cancer tissues were primarily distributed in distal intergenic regions and intronic regions. In gastric cancer tissues， compared to normal tissues， there was significant reprogramming of H3K27me3 modifications， characterized by a marked reduction in overall H3K27me3 signal intensity. The loss of 2 912 H3K27me3 signal peaks might lead to the up-regulation of 822 tumor-associated genes. Among them， 56 genes displayed the most significant up-regulation （fold change in signal intensity ≥2， P

ObjectiveTo investigate the expression and clinical significance of GATA-3 and H3K27me3 in Tibetan patients with bladder urothelial carcinoma (BUC).MethodsBUC and normal bladder tissues were collected retrospectively from January 2016 to December 2021 in the People's Hospital of Tibet Autonomous Region. The expression of GATA-3 and H3K27me3 in both tissues was detected by immunohistochemical method, and the clinical and pathological characteristics were statistically analyzed.ResultsA total of 70 patients with BUC were selected, including 51 males and 19 females, with an average age of (60.5±12.0) years. At the same time, 20 normal bladder tissue samples were collected during the same period. All cases were Tibetan patients. Immunohistochemistry results showed that the high expression rate of GATA-3 in BUC and normal bladder tissue was 70.0%(49/70) and 100%(20/20), respectively. High expression of GATA-3 was associated with male, low pathological grade, and non-invasive tissue(all P < 0.05). The high expression rate of H3K27me3 in BUC and normal bladder tissue was 45.7%(32/70) and 20.0%(4/20), respectively. High expression of H3K27me3 was only associated with male (P=0.011).ConclusionsThe expression of GATA-3 was down regulated in Tibetan BUC patients, and significantly down regulated with the increase of tumor grade, suggesting that GATA-3 may be involved in the occurrence and development of BUC and related to its malignancy, providing reference for clinical diagnosis and treatment as well as judging disease prognosis. The expression of H3K27me3 in Tibetan BUC patients was higher than that in normal bladder tissue, suggesting that H3K27me3 may be a new immune marker for diagnosis of BUC.

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Objective To investigate the expression and its significance of H3K27me3 in Tibetan patients with gastric cancer. Methods Clinical and pathological data were retrospectively collected from Tibetan patients with gastric cancer in the Tibet Autonomous Region People's Hospital from August 2019 to August 2021 and Tibetan non-gastric cancer patients during the same period. The expression of H3K27me3 in gastric cancer tissues, corresponding adjacent normal gastric mucosa and normal gastric mucosa of Tibetan patients with non-gastric cancer tissues was detected by immunohistochemical method, and the differences of H3K27me3 expression between gastric cancer patients with different clinical and pathological characteristics was compared. Results A total of 54 Tibetan gastric cancer patients and 55 Tibetan non-gastric cancer patients who met the inclusion and exclusion criteria were enrolled. H3K27me3 was localized in the nucleus, and the nucleus showed brownish-yellow granular staining when positively expressed. The high expression rate of H3K27me3 in gastric cancer tissues was significantly higher than that in adjacent normal gastric mucosa tissue[64.8%(35/54) vs. 29.6%(16/54), P < 0.001]and normal gastric mucosal tissue of Tibetan patients with non-gastric cancer[64.8%(35/54) vs. 34.5%(19/55), P=0.002], and the high expression rate of H3K27me3 in the adjacent normal gastric mucosa tissue was not significantly different from that in normal gastric mucosal tissue of Tibetan patients with non-gastric cancer(P=0.683). H3K27me3 expression in gastric cancer tissues was not related to gender, age, degree of differentiation, depth of invasion, maximum tumor size, Lauren's classification, lymph node metastasis, vascular and nerve invasion and TNM stage. Conclusions H3K27me3 is highly expressed in gastric cancer tissues of Tibetan people in Tibet. There is no significant difference in H3K27me3 positive expression cells in adjacent tissues and normal gastric mucosa. The significance of H3K27me3 in Tibetan patients with gastric cancer is uncertain and requires further investigations.

BACKGROUND: Valproic acid, as a histone deacetylase inhibitor, has the ability to promote osteogenic differentiation of cells, but the mechanism is still unclear. OBJECTIVE: To investigate the effect and mechanism of valproic acid on osteogenic differentiation of rat bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and identified by flow cytometry. CCK-8 assay was used to detect the effects of different concentrations of valproic acid on the proliferation of rat bone marrow mesenchymal stem cells, and determine the used concentration of valproic acid. After adding valproic acid, rat bone marrow mesenchymal stem cells were induced to osteogenic differentiation. Alkaline phosphatase staining was performed on day 7 of osteogenic induction culture, and alizarin red staining was performed on day 21 of osteogenic induction culture. Quantitative real-time polymerase chain reaction was used to detect the expression of osteogenesis-related genes and EZH2 on days 3 and 7 of osteogenic induction culture. The protein expression levels of Runt-related transcription factor 2, EZH2 and H3K27me3 were detected by western blot assay on day 5 of osteogenic induction culture. RESULTS AND CONCLUSION: (1) CCK-8 cell proliferation assay showed that bone marrow mesenchymal stem cell proliferation was significantly inhibited by increasing the concentration of valproic acid. (2) The results of alkaline phosphatase staining and alizarin red staining showed that valproic acid could increase the expression of alkaline phosphatase and enhance the ability of bone mineralization. (3) qRT-PCR results showed that valproic acid could increase the expression of osteogenesis-related genes and decrease the expression of EZH2 gene. (4) Western blot assay results showed that valproic acid could increase the expression of Runt-related transcription factor 2 protein and decrease the expression of EZH2 and H3K27me3 protein. (5) It is concluded that valproic acid can promote osteogenic differentiation of rat bone marrow mesenchymal stem cells by down-regulating EZH2 expression. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression and clinical significance of H3K27me3 in vascular hemangioma and malformation. Methods Paraffin-embedded tissue blocks from 244 patients with hemangioma and vascular malformation admitted in our hospital from January 2010 and June 2021 were collected. The expression levels of H3K27me3 and smooth muscle actin (SMA) in vascular endothelial and perivascular cells were detected by immunohistochemical SP assay, and its expression differences between hemangioma and vascular malformation was analyzed. Bioinformatics analysis was further applied to explore its potential role in anigodysplasia in combination of the results of SMA immunostaining. Receiver operating characteristic (ROC) curve was drawn to evaluate its diagnostic value in differentiation of hemangioma and vascular malformation. Results In vascular hemangioma, H3K27me3 expression was mainly restricted to vascular endothelial cells, with an expression rate of 50%~90%. However, in vascular malformations, it was expressed in vascular endothelial cells, with an expression rate of 30%~50%, and lost in perivascular cells. There were top 10 endothelium restricted genes derived from GSE21212 dataset based on concerning literatures. According to ChIP-seq data from ENCODE website, H3K27me3 was enriched in the SOX2 promoter of different endothelium lineages, and the molecular interaction of H3K27me3 and SOX2 may be associated with vessel development and differentiation. Conclusion The expression of H3K27me3 in vascular endothelial cells can be used as a candidate marker to distinguish hemangioma and vascular malformation. However, its expression in infantile hemangioma is immunologically heterogeneous.

OBJECTIVE: To explore the expression of histone H3K27me3 methyltransferase EZH2 in human lipomas, well-differentiated liposarcoma and dedifferentiated liposarcoma and the effect of the small molecule inhibitor of EZH2 enzyme activity GSK126 on human liposarcoma cell line SW872, and preliminary exploration Its possible mechanism. Methods: A total of 23 postoperative biopsy specimens from patients with lipoma, well-differentiated liposarcoma and dedifferentiated liposarcoma were screened, including 7 cases of lipoma, 9 cases of well-differentiated liposarcoma, and 7 cases of dedifferentiated liposarcoma. Histochemical staining detects the protein expression of EZH2. The SW872 liposarcoma cell line was cultured in vitro, the CCK-8 method was used to detect the inhibitory effect of different concentrations of GSK126 on cell survival, the flow cytometry method was used to detect cell apoptosis, and the Realtime PCR method was used to detect cell apoptosis and anti-apoptosis (Caspase-1)., Caspase-3, Caspase-7, Caspase-9, Bcl-2, Bag-3), angiogenesis (VEGF-α), dryness (CD133, CD44, CD24) related gene expression, Western blot detection of endoplasmic reticulum The expression levels of stress-related proteins Bip and ATF4. Results: The expression of EZH2 in dedifferentiated liposarcoma was higher than that in well-differentiated liposarcoma, and positive expression in benign liposarcoma was rare (all P<0.05). GSK126, an inhibitor of EZH2 enzyme activity, has a significant inhibitory effect on the survival of SW872 cells, and the apoptosis rate increases after administration (P<0.05). Apoptosis-related genes Caspase-1, Caspase-3, Caspase-7, and Caspase-9 The expression of both increased (all P<0.05), the expression of angiogenesis gene VEGF-α decreased (P<0.01), the expression of dryness gene CD133 decreased (P<0.01), and there was no significant difference in the expression of other genes. The expression of endoplasmic reticulum stress-related proteins Bip and ATF4 protein in the GSK126 group increased. Conclusion: The expression of EZH2 protein is negatively correlated with the degree of differentiation of liposarcoma cells. EZH2 is expected to become a biomarker of liposarcoma and a marker of malignancy. EZH2 inhibitors may become potential chemotherapeutic drugs for liposarcoma, and may play a role by enhancing endoplasmic reticulum stress. [ABSTRACT FROM AUTHOR]

Objective: To investigate the roles of histone H3K27me3 methylation and its regulatory enzymes JMJD3 and EZH2 in the differentiation of Th17 cells in ankylosing spondylitis (AS), to unveil their potential involvement in the pathogenesis of AS, and to provide new strategies and targets for the clinical treatment of AS by analyzing the methylation state of H3K27me3 and its interactions with Th17-related factors., Methods: A total of 84 AS patients (42 active AS patiens and 42 patients in the stable phase of AS) were enrolled for the study, while 84 healthy volunteers were enrolled as the controls. Blood samples were collected. Peripheral blood mononuclear cells were isolated. ELISA assay was performed to examine Th17 cells and the relevant cytokines IL-21, IL-22, and IL-17. The mRNA expressions of RORc , JAK2 , and STAT3 were analyzed by RT-PCR, the protein expressions of RORc, JAK2/STAT3 pathway protein, H3K27me3 and the relevant protease (EZH2 and JMJD3) were determined by Western blot. Correlation between H3K27me3, EZH2 and JMJD3 and the key signaling pathway molecules of Th cell differentiation was analyzed by Pearson correlation analysis., Results: The mRNA expressions of RORc , JAK2 , and STAT3 were significantly higher in the active phase group than those in the stable phase group ( P <0.05). The relative grayscale values of H3K27me3 and EZH2 in the active phase group were lower than those of the stable phase group, which were lower than those of the control group, with the differences being statistically significant ( P <0.05). The relative grayscale values of JMJD3, RORc, JAK2, pJAK2, STAT3, and pSTAT3 proteins were significantly higher in the active phase group than those in the stable phase group, which were higher than those in the control group (all P <0.05). The proportion of Th17 and the expression level of inflammatory factors in the active period group were higher than those in the other two groups (P<0.05). H3K27me3 was negatively correlated with RORc, JAK2, STAT3, and IL-17, JMJD3 was positvely correlated with JAK2, STAT3, and IL-17, and EZH2 was negatively correlated with JAK2, STAT3, and IL-17 (all P <0.05)., Conclusion: The low expression of H3K27me3 in AS is influenced by the gene loci JMJD3 and EZH2, which can regulate the differentiation of Th17 cells and thus play a role in the pathogenesis and progression of AS., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Office of Journal of Sichuan University (Medical Sciences).)

【目的】探索组蛋白H3K27me3 甲基转移酶Ezh2 在间充质干细胞内缺失对小鼠骨骼肌细胞分化及骨骼肌线粒体的影响。【方法】构建Ezh2 间充质干细胞条件性敲除小鼠（Ezh2F/FPrx1-Cre）。HE 染色、改良Gomori 三色染色及四氮唑还原酶染色（NADH-TR）法观察细胞形态及线粒体情况。电镜观察骨骼肌细胞和线粒体超微结构。Western Blot 法检测 6 周龄小鼠骨骼肌细胞中 Ezh2、H3K27me3 和骨骼肌分化相关基因 Myogenin、Myosin、Desmin 及线粒体蛋白CytC 的蛋白表达量。Realtime RT-qPCR 法检测骨骼肌分化相关基因和线粒体相关基因的表达情况。免疫组化染色检测骨骼肌中CytC 的表达量，ATP 含量测定试剂盒检测骨骼肌细胞内ATP 含量。HE和NADH-TR 染色观察2 周龄小鼠骨骼肌。检测6 周龄小鼠棕色脂肪中Ezh2 蛋白含量，电镜观察小鼠棕色脂肪中的线粒体。【结果】敲除组骨骼肌中 Ezh2 及H3K27me3 蛋白含量均明显降低。敲除组小鼠的骨骼肌肌纤维粗细不等，部分核内移，骨骼肌分化标志物 Myogenin 和 MhcIIb 的 mRNA 表达明显减少（均 P < 0.05），Myogenin 蛋白表达量减少，NADH-TR 和 CytC 免疫组化染色显示线粒体增加。电镜下敲除组骨骼肌肌原纤维粗细不等，细胞膜下及肌原纤维间线粒体数量明显增加，呈团块状聚集。线粒体无水肿，嵴无明显减少。敲除组线粒体相关基因CytC、Tfam、Pgc1α的mRNA 和CytC 蛋白表达升高（均P < 0.05），但ATP 含量明显降低（P < 0.05）。2 周龄小鼠骨骼肌纤维粗细不等，但 NADH 含量无差别。电镜下 6 周龄小鼠棕色脂肪细胞线粒体无数量增加或形态异常。【结论】Ezh2 在间充质干细胞阶段对小鼠骨骼肌细胞分化是必须的，并维持线粒体产生ATP 的功能。Ezh2 缺失小鼠骨骼肌细胞线粒体产生ATP 减少，线粒体数量代偿性增加。

To explore the effect of histone H3K27me3 methyltransferase Ezh2 on the differentiation of white, brown and beige adipocytes in mice. Ezh2 whole body knockout mice (Ezh2flox/floxCAGcre) were constructed and induced by intraperitoneal injection of tamoxifen at the age of 6 weeks. The same litter, same sex and same genotype pseudoinduction mice (intraperitoneal injection of corn oil) were used as control. After induction completion, the morphology of adipocytes was observed under light microscope, and the expression of H3K27me3, Ezh2 and Ucp1 in adipose tissue was detected by Western Blot method. Realtime PCR was used to detect the expression of adipose differentiation-related genes (Ppar酌, Adipoq and Fabp4), brown adipocyte markers (Ucp1, Cidea and Prdm16) and beige adipocyte markers (CD137, Tmem26 and Tbx1) in different parts of adipose tissues. The cold tolerance of knockout mice was tested, and obesity was induced by high fat diet. The weight gain, glucose tolerance and insulin sensitivity of mice were observed after induction. The protein content of Ezh2 and H3K27me3 in Ezh2 knockout mice decreased, the lipid droplets of brown adipocytes in interscapular region were significantly smaller than those in control group, and the gene and protein expression of Ucp1 were significantly higher than those in control group (P<0.05); the differentiation of white adipocytes in Ezh2 knockout mice was poor, the differentiation of beige adipocytes increased, and the expression of Ucp1 and Tbx1 genes in beige adipocytes increased(P<0.05). Ezh2 knockout mice were better able to tolerate cold stimulation and resist obesity and insulin resistance induced by a high-fat diet. Ezh2 promotes the differentiation of white adipocytes and inhibits the differentiation of brown and beige adipocytes in vivo. [ABSTRACT FROM AUTHOR]

Objective To explore the effects and mechanism of tripterine on the proliferation and apoptosis of human laryngeal carcinoma Hep-2 cells. Methods Human laryngeal carcinoma Hep-2 cells were treated with tripterine of different concentrations and in different days. The effects on cell proliferation were measured by trypan blue staining, CCK-8 method and crystal violet staining. The effects on cell apoptotis were observed by the fluorescence dye DAPI and flow cytometry. The protein expressions of EZH2 and H3K27me3 were detected by Western blot. Results Tripterine inhibited the proliferation of human laryngeal carcinoma Hep-2 cells in time-and dose-dependent manners ; IC50 was (3. 19±0. 83)μmol/L at 48 hours. Tripterine of 5 μmol/L could promote karyopyknosis and fragmentation of Hep-2 cell nucleus at 24 hours. The apoptosis rate was increased compared with that in control group (t = - 11.32, P <0.05). The protein expressions of EZH2 and H3K27me3 were downregulated. Conclusion Tripterine can significantly inhibit the proliferation and induce the apoptosis of human laryngeal carcinoma Hep-2 cells in vitro, which may be related to down-regulation of EZH2 and H3K27me3 expressions . [ABSTRACT FROM AUTHOR]

Objectives: Jmjd3 can promote the differentiation of brown adipocytes, but its role in the ultrastructure of lipid droplets and mitochondria, the two key thermogenic organelles, is still unclear. The aim of this study is to investigate the effects of histone H3K27me3 demethylase Jmjd3 deletion on lipid droplets and mitochondria in brown adipocytes in mice., Methods: Jmjd3 general knockout (Jmjd3 -/- ) mice and adipose tissue specific conditional knockout (Jmjd3 F/F Fabp4-Cre + ) mice were constructed.Brown adipose tissue (BAT) was taken from the back of Jmjd3 -/- and their littermates (Jmjd3 +/+ ) at the 19.5 days of embryo (E19.5). BAT was also taken from the back of 6-week-old Jmjd3 F/F Fabp4-Cre + and their littermates (Jmjd3 flox/+ or Jmjd3 +/+ Fabp4-Cre + ). The morphology of brown adipocytes was observed by light microscopy after HE staining. The ultrastructure of lipid droplets and mitochondria was observed by electron microscopy. Western blotting was used to detect the expression of H3K27me3, Jmjd3, and uncouple protein 1 (UCP1) in BAT. RT-qPCR was used to detect the expression of Jmjd3, peroxisome proliferator-activated receptor γ (PPARγ), fatty acid binding protein 4 (Fabp4), UCP1, PR domain-containing 16 (PRDM16), cell death-inducing DFFA-like effector a (CIDEa), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), mitochondrial cytochrome c oxidase subunit I (COX1), and cytochrome c (Cyt c). ATP content in the BAT was tested. The body temperature of Jmjd3 F/F Fabp4-Cre + and their control mice during cold stimulation was detected., Results: Two types of Jmjd3 knockout (Jmjd3 -/- and Jmjd3 F/F Fabp4-Cre + ) mice were smaller than their littermates. The expression of Jmjd3 mRNA ( P <0.05) and protein in BAT was significantly decreased, and the protein expression of H3K27me3 was increased, indicating that the knockout mice were successfully constructed. HE staining showed that lipid droplets in BAT of Jmjd3 -/- mice were significantly less than those in the control group. The results of electron microscopy showed that the area of brown adipocytes in Jmjd3 -/- mice was smaller ( P <0.05), the number of lipid droplets was less ( P <0.01), and the size of lipid droplets was not significantly different ( P >0.05). Mitochondrial edema and cristae rupture were observed in the Jmjd3 -/- group.The number and area of mitochondria were not affected (both P >0.05), the number of mitochondrial cristae was significantly less than that of the control group ( P <0.05). The protein expression of UCP1 of Jmjd3 -/- mice was decreased. The mRNA expression of UCP1, CIDEa, and PGC-1α in BAT of Jmjd3 -/- mice was significantly less than that of the control group (all P <0.05). ATP content in BAT of Jmjd3 -/- mice was decreased ( P <0.05). HE staining showed that lipid droplets in BAT of Jmjd3 F/F Fabp4-Cre + mice were larger than those in the control group. Electron microscopy showed that there was no significant difference in size of adipocytes between the 2 groups ( P >0.05). In the Jmjd3 F/F Fabp4-Cre + mice, lipid droplets were less ( P <0.05), but their diameters were larger ( P <0.001). Mitochondrial edema and cristae rupture were observed in the Jmjd3 F/F Fabp4-Cre + mice. The number of mitochondrial cristae in the Jmjd3 F/F Fabp4-Cre + mice was significantly less than that in the control group ( P <0.05). The protein expression of UCP1 in Jmjd3 F/F Fabp4-Cre + mice was decreased. The mRNA expression of UCP1, PRDM16, CIDEa, COX1, PGC-1α in BAT of Jmjd3 F/F Fabp4-Cre + mice was significantly less than that of the control group (all P <0.05). ATP content in BAT of Jmjd3 F/F Fabp4-Cre + mice was decreased ( P <0.05). In the Jmjd3 F/F Fabp4-Cre + mice, the body temperature was decreased more and the cold tolerance was poor ( P <0.05)., Conclusions: Jmjd3 promotes the differentiation of brown adipocytes by enhancing the formation of lipid droplets in mouse brown adipocytes and maintaining the normal morphology and function of mitochondria.